ABSTRACT
Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach ‘SMART-9N' and a version compatible rapid adapters available from Oxford Nanopore Technologies ‘Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.
ABSTRACT
Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.
ABSTRACT
Introdução O sequenciamento de genoma viral, projeções e visualizações por meio de modelos matemáticos, estatísticos e computacionais permitem acompanhar a disseminação de doenças infecciosas como a causada pela infecção pelo vírus SARS-CoV-2, a COVID-19. O monitoramento ativo e contínuo da evolução epidemiológica depende diretamente da vigilância atentando-se às variantes de preocupação, que podem ter maior transmissibilidade, virulência e letalidade que a linhagem original. Neste trabalho, apresentamos os resultados da genotipagem de amostras representativas distribuídas pelas Coordenadorias Regionais de Saúde do município de São Paulo. Os dados disponíveis para quase todo o ano de 2021 possuem informações como a data de coleta, data de primeiros sintomas, limiar Ct do exame de PCR, variante identificada e CEP do endereço de residência. Objetivo Na posse desses dados é possível analisar o padrão espaço-temporal da evolução da disseminação da COVID-19 no município de São Paulo por diferentes variantes, com o objetivo de determinar as regiões de surgimento de variantes de preocupação e estimar os padrões de mobilidade que permitam o espalhamento dessas variantes para diferentes locais. Método Os dados das amostras recebidas pela Secretaria de Saúde do Município de São Paulo são processados e completados com os resultados do sequenciamento genético por meio da técnica de PCR, determinando a variante identificada em cada uma dessas amostras. Depois, os dados passam por uma filtragem e correções de entradas, como as datas disponíveis e os CEPs. Em seguida, coordenadas geográficas dentro do município de São Paulo são obtidas, e mapas são construídos para mostrar o espalhamento da doença pelo município e a dominância de uma variante sobre a outra. Resultados O espalhamento da doença é visualizado por meio de mapas dinâmicos que permitem acompanhar o surgimento de variantes como a Gamma e a Delta em certas regiões do município, espalhando-se e dominando todo o território depois de um tempo. Com isso, foram identificadas as áreas mais suscetíveis e correlacionadas com os padrões de mobilidade urbana. Conclusão A vigilância da emergência e disseminação de variantes de preocupação permite a determinação de pontos chaves do comportamento viral e humano para determinar os locais mais suscetíveis a surtos e espalhamento de linhagens que são mais transmissíveis. Com isso, é possível estudar estratégias melhores para o combate não apenas da COVID-19, mas de outras doenças com padrões de transmissibilidade semelhantes. Ag. Financiadora FAPESP. Nr. Processo 2021/11953-5.
ABSTRACT
The first case of COVID-19 was detected in Brazil on 25 February 2020. We report and contextualize epidemiological, demographic and clinical findings for COVID-19 cases during the first 3 months of the epidemic. By 31 May 2020, 514,200 COVID-19 cases, including 29,314 deaths, had been reported in 75.3% (4,196 of 5,570) of municipalities across all five administrative regions of Brazil. The R0 value for Brazil was estimated at 3.1 (95% Bayesian credible interval = 2.4-5.5), with a higher median but overlapping credible intervals compared with some other seriously affected countries. A positive association between higher per-capita income and COVID-19 diagnosis was identified. Furthermore, the severe acute respiratory infection cases with unknown aetiology were associated with lower per-capita income. Co-circulation of six respiratory viruses was detected but at very low levels. These findings provide a comprehensive description of the ongoing COVID-19 epidemic in Brazil and may help to guide subsequent measures to control virus transmission.